In the conventional synthesis of peptides, there is known the active ester method in which the amino acid is firstly converted into an active ester thereof and then condensed with a second amino acid. For the activation of an amino acid, it is generally known that the amino acid may be converted into its active ester, its acid anhydride or its acid azide which is subsequently reacted with a second amino acid. It is known that the active ester method as well as the acid anhydride method and the azide method are unlikely to involve an undesired racemization of the amino acid employed. When the synthesis of peptides is performed according to the active ester method, the process of condensing an amino acid with a second amino acid usually includes two stages, that is, the first stage of preparing an active ester of the amino acid and the second stage of coupling the amino acid active ester so prepared with an amine component (namely, the second amino acid having the free amino group which is to be condensed with the activated carboxylate group of the amino acid active ester so that the amido linkage is formed between these two amino acid reactants). It has been reported that when the later stage, that is, the coupling stage is effected according to the known "backing off" method, the undesired racemization is unlikely to occur. Accordingly, if the stage of activating the amino acid can be effected substantially without involving the racemization, it is feasible to carry out the fragment condensation of the two amino acid reactants in a favorable way without bringing about the racemization of the amino acids throughout the overall process of synthesizing the peptides from amino acids.
In general, it is known that the active ester group which can be used to convert an amino acid into its active ester form includes p-nitrophenyl group, N-hydroxysuccinimido group and N-hydroxyphthalimido group. As one of the common methods for the preparation of the active esters of an amino acid, there may be mentioned such method in which the amino acid is reacted with N-hydroxysuccinimide (hereinafter sometime abbreviated as HOSu) in the presence of dicyclohexylcarbodiimide (usually abbreviated as DCC) as a dehydrating agent to form the N-hydroxysuccinimide ester of the amino acid (Anderson et al "Journal of American Chemical Society" 85, 3039 (1963)). This DCC method enjoys many advantages that the resulting amino acid N-hydroxysuccinimide ester is highly crystalisable (and hence readily purifiable) and highly reactive, and that the N-hydroxysuccinimide liberated in the coupling stage is soluble in water and hence can be removed from the reaction mixture merely by washing with water after the coupling reaction is completed. However, it is also known that the DCC method suffers from some drawbacks that the DCC used as the dehydrating agent and the amino acid N-hydroxysuccinimide ester prepared are both oleophilic in their nature so that the removal of the DCC from the reaction mixture containing the desired amino acid active ester prepared cannot be achieved merely by extracting with an organic solvent but needs troublesome operations for the isolation of the desired amino acid active ester product; that .beta.-alanine derivatives can be by-produced from the amino acid by Lossen-rearrangement under the action of DCC and HOSu; and that when the DCC method is applied to asparagine, glutamine or such another amino acids containing once an acid amido group in the molecule thereof, a nitrile derivative can undesirably by by-produced therefrom by dehydration under the action of DCC.
Further known methods for the preparation of the N-hydroxysuccinimide ester of an amino acid include such method in which trifluoroacetic acid anhydride is used and reacted with N-hydroxysuccinimide to form the N-hydroxysuccinimide ester of trichloroacetic acid which is then reacted with an amino acid to produce the active ester of the amino acid through the ester-interchange reaction (Stepanoya et al, "J. Gen. Chem." (USSR) 45, 2451 (1976)). When this prior art method is applied to N-t-butoxycarbonylglycine (Boc-Gly), the reactions involved therein may be shown by the following scheme: ##STR1## With this prior art method, however, the trichloroacetic acid N-hydroxysuccinimide ester obtained as the intermediate must be prepared just before its use and hence can neither be isolated nor be purified, so that the active ester of the amino acid obtained using the trichloroacetic acid N-hydroxysuccinimide ester needs repeated purifications. Besides, the reaction of preparing the intermediate trichloroacetic acid N-hydroxysuccinimide ester has to be carried out under absolutely anhydrous conditions, which provides a disadvantage, too.
According to Groos et al ("Agnew. Chem. Internt." Edit. 6, 570 (1967)), N-hydroxysuccinimide potassium salt is reacted with phosgene to prepare N-hydroxysuccinimide chloroformate which is then reacted with an N-protected amino acid in the presence of triethylamine to give a mixed acid anhydride. The mixed acid anhydride so prepared is subsequently heated to yield the N-hydroxysuccinimide active ester of the N-protected amino acid. When this method of Groos et al is applied to N-benzyloxycarbonylphenylalanine (Z-Phe), the reactions involved therein are shown by the following scheme: ##STR2##
The method of Groos et al, however, can suffer from some disadvantages that the operations required in this method are complicated and that there involves a risk that the undesired racemization of the amino acids employed could take place due to the effect of chloride ions which would be formed during the reactions.
According to Ito ("Bull. Chem. Soc. Jpn." 47, 471 (1974)), there is proposed a method of activating an organic acid in which an organic acid such as benzoic acid is activated by reacting with an asymmetrical carbonate derivative, particularly ethyl 2-ethoxycarbonyloxyimino-2-cyanoacetate in the presence of diethylamine, as illustrated by the following scheme: ##STR3##
With this method of Ito, it is to be noticed that two different esters can be formed concurrently due to the assymmetry of the carbonate derivative employed as the activating reagent, and that one of the two esters formed is the activated ester as desired but the other is not the activated ester. According to the method of Ito, it is also possible to employ ethyl 2-(p-nitrophenyloxy)-carbonyloxyimino-2-cyanoacetate as the activating assymmetrical carbonate derivative. When using this particular carbonate compound, an amino acid can be converted into its active ester in a yield of 50 to 60%. When this procedure according to the method of Ito is applied to N-benzyloxycarbonylphenylalanine (Z-Phe), the reactions involved therein may be shown by the following scheme: ##STR4##
With this procedure, however, there is not obtained the desired active ester of the amino acid which is unlikely to involve racemization of the amino acid.
According to Glatthard et al ("Helv. Chem. Acta." 46, 795 (1963)), there is proposed a method for preparing an active ester of an amino acid in a favorable yield by reacting an amino acid with a diphenyl carbonate derivative which is formed by reaction of a certain phenol derivative with phosgene. When this diphenyl carbonate derivative is employed as the reagent of activating the amino acid, however, there involves a risk that the amino acid can undergo racemization in the stage of the aminolysis, that is, the stage where the activated amino acid ester is coupled with the amine component (i.e. the second amino acid) to produce the amido linkage.